Mixed population of cells deriving from adipose tissue and methods of isolating and using the same

ABSTRACT

Adipose tissue-derived stromal cells and methods of isolating and using the same. In at least one embodiment of isolated adipose tissue-derived stromal cells of the present disclosure, the cells are isolated by performing adipose tissue resection or suction on a mammalian patient, dissecting tissue obtained from said tissue resection or suction and dissociating said tissue into a cell suspension, removing adipocytes from the cell suspension, culturing the adipocyte-depleted cell suspension in EGM-2-MV media, and isolating adipose tissue-derived stromal cells secreting vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and granulocyte-colony stimulating factor (G-CSF).

PRIORITY

The present application is related to, claims the priority benefit of,and is a continuation application of, U.S. patent application Ser. No.13/363,399, filed Feb. 1, 2012, which is related to, claims the prioritybenefit of, and is a divisional application of, U.S. patent applicationSer. No. 13/279,222, filed Oct. 21, 2011, which is related to, claimsthe priority benefit of, and is a divisional application of, U.S. patentapplication Ser. No. 12/569,887, filed Sep. 29, 2009 and issued as U.S.Pat. No. 8,067,234 on Nov. 29, 2011, which is related to, claims thepriority benefit of, and is a divisional application of, U.S. patentapplication Ser. No. 10/508,223, filed Jun. 23, 2005, which is relatedto, claims the priority benefit of, and is a U.S. §371 National StageApplication of, International Patent Application Serial No.PCT/US2003/008582, filed Mar. 19, 2003, which is related to, and claimsthe priority benefit of, U.S. Patent Application Ser. No. 60/365,498,filed Mar. 19, 2002. The contents of each of these applications arehereby incorporated by reference in their entirety into this disclosure.

BACKGROUND

The present disclosure relates to the fields of cardiology,vascularization, and molecular biology. More specifically, methods areprovided for isolating stem cells from adipose tissue, optionallyinducing the stem cells to secrete endogenous or exogenously providedgrowth factors, and re-administering such stem cells to a patient fortherapeutic benefit.

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisdisclosure pertains. Each of these references is incorporated herein asthough set forth in full.

Coronary artery disease (CAD) is a major cause of morbidity andmortality, requiring bypass surgery or angioplasty in almost 1,000,000patients/year in the USA. While some of these patients form collateralvessels as alternative pathways for blood supply, thus ameliorating orpreventing ischemic myocardial damage, many do not form the vascularnetworks to sufficiently compensate for the loss of the original bloodsupply. Accordingly, many patients could be helped by the development ofcompositions and methods which would accelerate natural processes ofpost-natal collateral vessel formation. Such approaches are broadlyreferred to as “therapeutic angiogenesis”, and encompass bothangiogenesis (which strictly speaking refers to capillary sprouting) andarteriogenesis (the maturation and enlargement of existing vessels)(Isner J. M. and Asahara T., J Clin Invest. (1999) 103:1231-1236; vanRoyen N. et al., Cardiovasc Res. (2001) 49:543-553.)

An emerging therapeutic approach is the use of stem and progenitor celltransplantation to improve angiogenesis. Endothelial progenitor cells(EPCs) are cells present in bone marrow or peripheral blood whichco-express stem and progenitor cell markers like CD34 or AC133, as wellas endothelial markers like VE-Cadherin and VEGF-Receptor-2 (KDR) (RafiiS., J Clin Invest. (2000) 105:17-19). EPCs and hematopoietic stem cells(HSCs) are thought to be derived from a common “hemangioblast” precursor(Ribatti D. et al., J Hematother Stem Cell Res. (2000) 9:13-19; Choi K.,J Hematother Stem Cell Res. (2002) 11:91-101; Eichmann A. et al., JHematother Stem Cell Res. (2002) 11:207-214). Interestingly, the cellsurface marker CD34 is only found on either hematopoieticstem/progenitor cells or endothelial cells (Rafii S., J Clin Invest.(2000) 105:17-19), which may be a reflection of the common origin ofthese two cell lineages. In addition to the shared “hemangioblastic”ancestry between HSCs and endothelial cells, HSCs have also beensuggested to trans-differentiate into either endothelial progenitorcells or mature endothelial cells (Kang H. J. et al., Br J Haematol.(2001) 113:962-969; Quirici N. et al., Br J Haematol. (2001)115:186-194; Gehling U. M. et al., Blood. (2000) 95:3106-3112). Therecent discovery of circulating smooth muscle progenitor cells, and thepotential of HSCs to differentiate into smooth muscle cells (Sata M. etal., Nat Med. (2002) 8:403-409; Simper D. et al., Circulation (2002)106:1199-1204) may suggest yet another novel and intriguing link betweenthe hematopoietic and vascular cell lineages, now in the context ofsmooth muscle cells.

Animal studies using hindlimb ischemia or myocardial ischemia models inimmune deficient rodents have demonstrated that transplantation of about10⁶ peripheral blood derived

EPCs (Kawamoto A. et al., Circulation (2001) 103:634-637; Kalka C. etal., Proc Natl Acad Sci USA (2000) 97:3422-3427) can result in increasedangiogenesis. Remarkably, labeled peripheral blood derived EPCs appearto home preferentially to ischemic areas and incorporate into foci ofneovascularization (Kawamoto A. et al., Circulation (2001) 103:634-637;Kalka C. et al., Proc Natl Acad Sci USA (2000) 97:3422-3427). Inaddition to the above-mentioned studies on peripheral blood-derivedcells, EPCs derived from bone marrow, unpurified bone marrow mononuclearcells, and HSCs have also been shown to enhance angiogenesis or showendothelial differentiation in vivo in a variety of animal models ofischemia (Kocher A. A. et al., Nat Med. (2001) 7:430-436; Shintani S. etal., Circulation (2001) 103:897-903; Fuchs S. et al., J Am Coll Cardiol(2001) 37:1726-1732; Kamihata H. et al., Circulation (2001)104:1046-1052; Orlic D. et al., Proc Natl Acad Sci USA (2001)98:10344-10349; Jackson K. A. et al., J Clin Invest. (2001)107:1395-1402).

Transplantation of hematopoietic stem cells (HSCs) into patients withmyelodysplastic disorders or following myeloablative radiochemotherapyis the most widespread application of stem cell therapy. HSCs arecharacterized by surface markers like CD34, and constitute less than0.5-1% of the bone marrow (Gunsilius E. et al., Biomed Pharmacother.(2001) 55:186-194), which is currently the primary source oftransplantable HSCs in the clinical setting. The limited availability ofHLA-compatible siblings (less than 30%) (Tabbara I. A. et al., ArchIntern Med. (2002) 162:1558-1566) has resulted in frequent use ofnon-HLA-compatible siblings as donors. Although recently there has beensome success in the reduction of complications following allogeneictransplantation of HSCs, chronic graft versus host disease andengraftment failure of allogeneic cells remains a significant clinicalproblem (Tabbara I. A. et al., Arch Intern Med. (2002) 162:1558-1566).Autologous stem cell transplantation circumvents these complications,however, autologous cells from the bone marrow or peripheral blood maybe contaminated by malignant cells (Hahn U. and To L. B., In: SchindhelmK., Nordon R. eds. Ex vivo Cell Therapy. San Diego, Calif.: AcademicPress; (1999) 99-126).

While the concept of using autologous peripheral blood derived EPCs inpatients seems attractive, based on animal studies, one would need 12liters of blood from a patient to isolate enough cells to achieve apro-angiogenic effect (Iwaguro H. et al., Circulation. (2002)105:732-738). This amount of blood is not readily available in aclinical setting. Human studies that have used bone marrow celltransplantation in ischemic patients (Strauer B. E. et al., Dtsch MedWochenschr. (2001) 126:932-938; Tateishi-Yuyama E. et al., Lancet.(2002) 360:427-435) suggest that human angiogenic cell therapy requiresat least cell numbers of 10⁷ to 10⁹, depending on the degree of stemcell purity as well as the optimal delivery method.

The discovery of pluripotent cells in the adipose tissue (Zuk P. A. etal., Tissue Engineering. (2001) 7:211-228) has revealed a novel sourceof cells that may be used for autologous cell therapy to regeneratetissue. The pluripotent cells reside in the “stromal” or “non-adipocyte”fraction of the adipose tissue; they were previously considered to bepre-adipocytes, i.e. adipocyte progenitor cells, however recent datasuggests a much wider differentiation potential. Zuk et al. were able toestablish differentiation of such subcutaneous human adipose stromalcells (ASCs) in vitro into adipocytes, chondrocytes and myocytes (Zuk P.A. et al., Tissue Engineering. (2001) 7:211-228). These findings wereextended in a study by Erickson et al., which showed that human ASCscould differentiate in vivo into chondrocytes (Erickson et al., BiochemBiophys Res Commun. (2002) 290:763-769) following transplantation intoimmune-deficient mice. More recently, it was demonstrated that humanASCs were able to differentiate into neuronal cells (Safford K. M. etal., Biochem Biophys Res Commun. (2002) 294:371-379).

Given the many applications of stem cell therapy, a need exists in theart for providing a more abundant and practical source for such stemcells, and for enhancing the potential therapeutic benefit and route ofadministration of these cells.

BRIEF SUMMARY

In accordance with the present disclosure, isolated, adipose tissuederived stromal cells are provided. The cells of the present disclosuremay be induced to express at least one characteristic of a variety ofcell types including but not limited to cardiac cells, endothelialcells, smooth muscle cells, dopaminergic neuronal cells, hematopoieticcells, or hepatic cells. Further, the cells may be induced to express agrowth factor including but not limited to vascular endothelial growthfactor (VEGF), hepatocyte growth factor (HGF), granulocyte-colonystimulating factor (G-CSF), and basic fibroblast growth factor (bFGF),with or without subsequent cell culture.

Another object of the present disclosure is to provide a method ofpromoting angiogenesis, cardiomyogenesis, or regeneration ofhematopoietic cells in which the cells of the present disclosure aredelivered to a patient. The cells may be delivered by methods whichinclude but are not limited to retrograde coronary venous infusion,retrograde delivery to other tissues, direct injection into targettissue, intra-arterial or intra-coronary infusion via a catheter, andsystemic intravenous administration.

Another object of the disclosure is a method for isolating adiposederived stem cells, and optionally culturing the cells in a medium ofinterest. As a further option, these cells may be exposed to a receptorligand cocktail including but not limited to at least one of VEGF, LIF,bFGF, IGF1, IGF2, HGF, cardiotrophin, myotrophin, nitric oxide synthase3, tumor necrosis factor alpha, tumor necrosis factor beta, fibroblastgrowth factor, pleotrophin, endothelin, and angiopoietin.

In yet a further embodiment of the present disclosure, the adiposederived stromal cells may comprise an exogenous nucleic acid encoding aprotein of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show phase contrast micrographs of sub-confluent (1A) andconfluent (1B) cultures of human adipose stromal cells grown in EGM2MVmedia, at 250× magnification.

FIG. 2 is a graph showing growth of human subcutaneous adipose stromalcells.

FIG. 3 shows flow cytometric analysis of fresh human subcutaneousadipose stromal cells.

FIG. 4 shows flow cytometric analysis of Sca-1 expression (green) andthe corresponding isotype control (pink.)

FIG. 5 shows flow cytometric analysis of CD-34 and VE-cadherinexpression on plated adipose stromal cells.

FIGS. 6A-B show human aortic endothelial cells (6A) and human adiposestromal cells (6B) plated overnight on Matrigel.

FIG. 7 shows smooth-muscle alpha-actin staining on adipose stromalcells.

FIG. 8 is a graph which shows secretion of growth factors VEGF, HGF, andG-CSF, presented as mean+/−standard error of mean of pg/10⁶ cells.

FIG. 9 is a micrograph of GFP expression on porcine adipose stromalcells. The fluorescent image is overlaid 10 on a phase contrast image.

FIG. 10 shows flow cytometric analysis of murine adipose stromal cellsgated on CD45(−) cells. The R3 cells are an ASC Sca-1(+)CD45(−)c-kit(−)population, while the R4 cells are Sca-1(−)CD45(−)c-kit(−).

FIGS. 11A-E show a series of micrographs (320×) taken following 12 daysof culture of Sca-1(+)CD45(−)c-kit(−) cells from muscle tissue andadipose tissue, which shows adipocyte-like differentiation (Oil-Red-0stain, 11A and 11D) or neuron-like differentiation (phase-contrast in11B and 11E) when grown in the respective differentiation media. Panel11C shows staining for the neuronal tau-protein (red) and nucleistaining (DAPI, blue) with confocal microscopy.

FIGS. 12A-F are micrographs of porcine myocardium tissue, showing thedistribution of BrdU labeled cells (marked with arrows) followingadministration by retrograde coronary venous delivery (12A-D) and bydirect injection (12E-F).

FIGS. 13A-B show laser Doppler imaging of the mouse hindlimb, whichdemonstrates that the Rac −/− mouse (13A) has persistent markedimpairment of the ischemic leg (right leg) perfusion, whereas thewild-type mouse (13B) shows significant restoration of blood perfusionto the ischemic leg (right leg.) High or normal blood perfusion isdepicted in red, and low or absent perfusion is shown in yellow andgreen.

FIG. 14 shows the ratio of perfusion to the ischemic leg to that of thenon-ischemic leg measured by laser Doppler imaging in wild-type and Rac2−/− mice.

FIGS. 15A-B show a representative depiction of limb necrosis on day 10in a media injected mouse (15A) and an adipose stromal cell injectedmouse (15B), which shows that adipose stromal cell injection canattenuate ischemia-induced necrosis.

FIG. 16 is a graph showing that on day 10, adipose stromal cell treatedanimals had a significantly reduced degree of limb necrosis whencompared to the control media injected mice.

DETAILED DESCRIPTION

Stem cell therapies are expected to someday provide a wide range oftreatment options for various diseases. However the difficultiesassociated with obtaining stem cells in therapeutic quantities hastroubled the scientific community. As described above, stem cells fromblood and/or bone marrow cannot be isolated in sufficient quantity fortherapy without significant cell expansion. Further, it is difficult tofind an HLA matched donor, and allogenic transplants pose a high risk ofcomplications.

New methods of deriving stem cells from adipose tissue provide anexcellent solution to these challenges. The methods disclosed hereinwill demonstrate that 10⁵ to 10⁶ cells that are obtained from 5-10 gramsof subcutaneous tissue can be expanded 50-fold in approximately oneweek. Considering that the simple outpatient procedure of liposuctioncan often yield 1 liter of fat tissue even in non-obese patients, ASCcell numbers of 10⁸ to 10⁹ can be isolated from an individual withlittle or no expansion.

Furthermore, the methods disclosed herein demonstrate ways to inducethese adipose stromal cells to secrete useful growth factors which canpromote new tissue growth. For example, ASC's can be induced to secreteVascular Endothelial Growth Factor (VEGF), which is known to be aneffective inducer of angiogenesis.

Methods provided herein combine efficient isolation of therapeuticquantities of stem cells with inducing these stem cells to secretegrowth factors such as VEGF. These cells can then be administered to apatient to promote vascularization and tissue growth in areas ofischemic damage.

I. Definitions

The following definitions are provided to facilitate an understanding ofthe present disclosure:

The term “autologous” implies identical nuclear genetic identity betweendonor cells or tissue and those of the recipient.

The term “adipose stromal cells” refers to the “non-adipocyte” fractionof adipose tissue. The cells can be fresh, or in culture. Adiposestromal cells contain pluripotent cells, which have the ability todifferentiate into cell types including but not limited to adipocytes,cardiomyocytes, endothelial cells, hematopoietic cells, hepatic cells,chondrocytes, osteoblasts, neuronal cells, and myotubes. “Adipose stemcells” are cells within the adipose stromal fraction which exhibit astem cell phenotype, such as CD45−/Sca-1+/c-kit- or CD45−/CD34+/c-kit-.

“Therapeutic quantities of stromal cells” or “therapeutic quantities ofstem cells” refers to the minimum amount of stem cells which willproduce a desired therapeutic effect. For example, a therapeuticquantity of VEGF secreting stem cells is the quantity which will producetherapeutically beneficial levels of angiogenesis, when administered toa patient.

“Therapeutic angiogenesis” or “therapeutic vascular growth” includes butis not limited to angiogenesis (such as capillary sprouting) andarteriogenesis (such as the maturation and enlargement of existingvessels.)

The term “regeneration” or “tissue regeneration” includes, but is notlimited to the growth, generation, or reconstruction of new cells typesor tissues from the ASCs of the instant disclosure. These cells types ortissues include but are not limited to endothelial cells,cardiomyocytes, hematopoietic cells, hepatic cells, adipocytes,chondrocytes, osteoblasts, neuronal cells, and myotubes.Cardiomyogenesis refers to cardiac tissue regeneration orreconstruction. Tissue regeneration also includes bone marrowrepopulation using the ASCs of the present disclosure, which expresshematopoietic lineage cell marker(s).

“Growth factors” are molecules which promote tissue growth, cellularproliferation, vascularization, and the like. These include, but are notlimited to Vascular Endothelial Growth Factors A, B, C, D, and E (VEGF),Placental Growth Factor, Hepatocyte Growth Factor (HGF),Granulocyte-Colony Stimulating Factor, Granulocyte-macrophage ColonyStimulating Factor, Macrophage Colony Stimulating Factor, MonocyteChemotactic Factor, TGF family, FGF family, pleiotrophin, endothelin,angiopoietins, and so forth.

“Multipotent” implies that a cell is capable, through its progeny, ofgiving rise to several different cell types found in the adult animal.

“Pluripotent” implies that a cell is capable, through its progeny, ofgiving rise to all the cell types which comprise the adult animalincluding the germ cells. Both embryonic stem and embryonic germ cellsare pluripotent cells under this definition.

The term “transgenic” animal or cell refers to animals or cells whosegenome has been subject to technical intervention including theaddition, removal, or modification of genetic information. The term“chimeric” also refers to an animal or cell whose genome has modified.

The term “cultured” as used herein in reference to cells can refer toone or more cells that are undergoing cell division or not undergoingcell division in an in vitro environment. An in vitro environment can beany medium known in the art that is suitable for maintaining cells invitro, such as suitable liquid media or agar, for example. Specificexamples of suitable in vitro environments for cell cultures aredescribed in Culture of Animal Cells: a manual of basic techniques (3rdedition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells: alaboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A.Leinwand (eds.), Cold Spring Harbor Laboratory Press; and Animal Cells:culture and media, 1994, D. C. Darling, S. J. Morgan John Wiley andSons, Ltd.

The term “cell line” as used herein can refer to cultured cells that canbe passaged at least one time without terminating. The presentdisclosure relates to cell lines that can be passaged at least 1, 2, 5,10, 15, 20, 30, 40, 50, 60, 80, 100, and 200 times. Cell passaging isdefined hereafter.

The term “suspension” as used herein can refer to cell cultureconditions in which cells are not attached to a solid support. Cellsproliferating in suspension can be stirred while proliferating usingapparatus well known to those skilled in the art.

The term “monolayer” as used herein can refer to cells that are attachedto a solid support while proliferating in suitable culture conditions. Asmall portion of cells proliferating in a monolayer under suitablegrowth conditions may be attached to cells in the monolayer but not tothe solid support. Preferably less than 15% of these cells are notattached to the solid support, more preferably less than 10% of thesecells are not attached to the solid support, and most preferably lessthan 5% of these cells are not attached to the solid support.

The term “plated” or “plating” as used herein in reference to cells canrefer to establishing cell cultures in vitro. For example, cells can bediluted in cell culture media and then added to a cell culture plate,dish, or flask. Cell culture plates are commonly known to a person ofordinary skill in the art. Cells may be plated at a variety ofconcentrations and/or cell densities.

The term “cell plating” can also extend to the term “cell passaging.”Cells of the present disclosure can be passaged using cell culturetechniques well known to those skilled in the art. The term “cellpassaging” can refer to a technique that involves the steps of (1)releasing cells from a solid support or substrate and disassociation ofthese cells, and (2) diluting the cells in media suitable for furthercell proliferation. Cell passaging may also refer to removing a portionof liquid medium containing cultured cells and adding liquid medium tothe original culture vessel to dilute the cells and allow further cellproliferation. In addition, cells may also be added to a new culturevessel which has been supplemented with medium suitable for further cellproliferation.

The term “proliferation” as used herein in reference to cells can referto a group of cells that can increase in number over a period of time.

The term “permanent” or “immortalized” as used herein in reference tocells can refer to cells that may undergo cell division and double incell numbers while cultured in an in vitro environment a multiple numberof times until the cells terminate. A permanent cell line may doubleover 10 times before a significant number of cells terminate in culture.Preferably, a permanent cell line may double over 20 times or over 30times before a significant number of cells terminate in culture. Morepreferably, a permanent cell line may double over 40 times or 50 timesbefore a significant number of cells terminate in culture. Mostpreferably, a permanent cell line may double over 60 times before asignificant number of cells die in culture.

The term “precursor cell” or “precursor cells” as used herein can referto a cell or cells used to establish cultured mammalian cells or acultured mammalian cell line. A precursor cell or cells may be isolatedfrom nearly any cellular entity.

The term “reprogramming” or “reprogrammed” as used herein can refer tomaterials and methods that can convert a cell into another cell havingat least one differing characteristic. Also, such materials and methodsmay reprogram or convert a cell into another cell type that is nottypically expressed during the life cycle of the former cell. Forexample, (1) a non-totipotent cell can be reprogrammed into a totipotentcell; (2) a precursor cell can be reprogrammed into a cell having amorphology of an EG cell; and (3) a precursor cell can be reprogrammedinto a totipotent cell. An example of materials and methods forconverting a precursor cell into a totipotent cell having EG cellmorphology is described hereafter.

The term “isolated” as used herein can refer to a cell that ismechanically separated from another group of cells. Examples of a groupof cells are a developing cell mass, a cell culture, a cell line, and ananimal.

The term “non-embryonic cell” as used herein can refer to a cell that isnot isolated from an embryo. Non-embryonic cells can be differentiatedor nondifferentiated. Non-embryonic cells can refer to nearly anysomatic cell, such as cells isolated from an ex utero animal. Theseexamples are not meant to be limiting.

The term “differentiated cell” as used herein can refer to a precursorcell that has developed from an unspecialized phenotype to a specializedphenotype. For example, embryonic cells can differentiate into anepithelial cell lining the intestine. Materials and methods of thepresent disclosure can reprogram differentiated cells into totipotentcells. Differentiated cells can be isolated from a fetus or a live bornanimal, for example.

The term “undifferentiated cell” as used herein can refer to a precursorcell that has an unspecialized phenotype and is capable ofdifferentiating. An example of an undifferentiated cell is a stem cell.

The term “asynchronous population” as used herein can refer to cellsthat are not arrested at any one stage of the cell cycle. Many cells canprogress through the cell cycle and do not arrest at any one stage,while some cells can become arrested at one stage of the cell cycle fora period of time. Some known stages of the cell cycle are G1, S, G2, andM. An asynchronous population of cells is not manipulated to synchronizeinto any one or predominantly into any one of these phases. Cells can bearrested in the M stage of the cell cycle, for example, by utilizingmultiple techniques known in the art, such as by colcemid exposure.Examples of methods for arresting cells in one stage of a cell cycle arediscussed in WO 97/07669, entitled “Quiescent Cell Populations forNuclear Transfer”.

The terms “synchronous population” and “synchronizing” as used hereincan refer to a fraction of cells in a population that are within a samestage of the cell cycle. Preferably, about 50% of cells in a populationof cells are arrested in one stage of the cell cycle, more preferablyabout 70% of cells in a population of cells are arrested in one stage ofthe cell cycle, and most preferably about 90% of cells in a populationof cells are arrested in one stage of the cell cycle. Cell cycle stagecan be distinguished by relative cell size as well as by a variety ofcell markers well known to a person of ordinary skill in the art. Forexample, cells can be distinguished by such markers by using flowcytometry techniques well known to a person of ordinary skill in theart. Alternatively, cells can be distinguished by size utilizingtechniques well known to a person of ordinary skill in the art, such asby the utilization of a light microscope and a micrometer, for example.In a preferred embodiment, cells are synchronized by arresting them(i.e., cells are not dividing) in a discreet stage of the cell cycle.

An “exogenous nucleic acid” as used herein refers to any nucleic acidwhich is introduced into the ASCs of the present disclosure, and encodesa protein of interest. Specific exogenous nucleic acids encode proteinswhich include without limitation a Vascular Endothelial Growth Factor A,B, C, D, and E (VEGF), bFGF, IGF1, IGF2, Hepatocyte Growth Factor (HGF),Placental Growth Factor, cardiotrophin, myotrophin, nitric oxidesynthases 1, 2, or 3, Granulocyte-Colony Stimulating Factor,Granulocyte-macrophage Colony Stimulating Factor, Macrophage ColonyStimulating Factor, Monocyte Chemotatic Factor, TGF family, FGF family,pleiotrophin, endothelin, angiopoietins, and genes promotingdifferentiation along pre-determined pathways.

The term “modified nuclear DNA” as used herein can refer to a nucleardeoxyribonucleic acid sequence of a cell of the present disclosure thathas been manipulated by one or more recombinant DNA techniques. Examplesof recombinant DNA techniques are well known to a person of ordinaryskill in the art, which can include (1) inserting a DNA sequence fromanother organism (e.g., a human organism) into target nuclear DNA, (2)deleting one or more DNA sequences from target nuclear DNA, and (3)introducing one or more base mutations (e.g., site-directed mutations)into target nuclear DNA. Cells with modified nuclear DNA can be referredto as “transgenic cells” or “chimeric cells” for the purposes of thepresent disclosure. The phrase “modified nuclear DNA” may also encompass“corrective nucleic acid sequence(s)” which replace a mutated nucleicacid molecule with a nucleic acid encoding a biologically active,phenotypically normal polypeptide. The constructs utilized to generatemodified nuclear DNA may optionally comprise a reporter gene encoding adetectable product.

As used herein, the terms “reporter,” “reporter system”, “reportergene,” or “reporter gene product” shall mean an operative genetic systemin which a nucleic acid comprises a gene that encodes a product thatwhen expressed produces a reporter signal that is a readily measurable,e.g., by biological assay, immunoassay, radioimmunoassay, or bycalorimetric, fluorogenic, chemiluminescent or other methods. Thenucleic acid may be either RNA or DNA, linear or circular, single ordouble stranded, antisense or sense polarity, and is operatively linkedto the necessary control elements for the expression of the reportergene product. The required control elements will vary according to thenature of the reporter system and whether the reporter gene is in theform of DNA or RNA, but may include, but not be limited to, suchelements as promoters, enhancers, translational control sequences, polyA addition signals, transcriptional termination signals and the like.

“Selectable marker” as used herein refers to a molecule that whenexpressed in cells renders those cells resistant to a selection agent.Nucleic acids encoding selectable marker may also comprise such elementsas promoters, enhancers, translational control sequences, poly Aaddition signals, transcriptional termination signals and the like.Suitable selection agents include antibiotic such as kanamycin,neomycin, and hygromycin.

Methods and tools for insertion, deletion, and mutation of nuclear DNAof mammalian cells are well-known to a person of ordinary skill in theart. See, Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989,Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press;U.S. Pat. No. 5,633,067, “Method of Producing a Transgenic Bovine orTransgenic Bovine Embryo,” DeBoer et al., issued May 27, 1997; and U.S.Pat. No. 5,612,205, “Homologous Recombination in Mammalian Cells,” Kayet al., issued Mar. 18, 1997. These methods include techniques fortransfecting cells with foreign DNA fragments and the proper design ofthe foreign DNA fragments such that they effect insertion, deletion,and/or mutation of the target DNA genome.

The adipose stromal cells defined herein can be altered to harbormodified nuclear or cytoplasmic DNA.

Examples of methods for modifying a target DNA genome by insertion,deletion, and/or mutation are retroviral or adeno-associated viralinsertion, artificial chromosome techniques, gene insertion byelectroporation, sonoporation, or chemical methods, random insertionwith tissue specific promoters, homologous recombination, genetargeting, transposable elements, and/or any other method forintroducing foreign DNA. Other modification techniques well known to aperson of ordinary skill in the art include deleting DNA sequences froma genome, and/or altering nuclear DNA sequences. Examples of techniquesfor altering nuclear DNA sequences are site-directed mutagenesis andpolymerase chain reaction procedures. Therefore, the present disclosurerelates in part to mammalian cells that are simultaneously totipotentand transgenic. Such transgenic and totipotent cells can serve as nearlyunlimited sources of donor cells for production of cloned transgenicanimals.

The term “recombinant product” as used herein can refer to the productproduced from a DNA sequence that comprises at least a portion of themodified nuclear DNA. This product can be a peptide, a polypeptide, aprotein, an enzyme, an antibody, an antibody fragment, a polypeptidethat binds to a regulatory element (a term described hereafter), astructural protein, an RNA molecule, and/or a ribozyme, for example.These products are well defined in the art.

The term “promoters” or “promoter” as used herein can refer to a DNAsequence that is located adjacent to a DNA sequence that encodes arecombinant product. A promoter is preferably linked operatively to anadjacent DNA sequence. A promoter typically increases an amount ofrecombinant product expressed from a DNA sequence as compared to anamount of the expressed recombinant product when no promoter exists. Apromoter from one organism can be utilized to enhance recombinantproduct expression from a DNA sequence that originates from anotherorganism. In addition, one promoter element can increase an amount ofrecombinant products expressed for multiple DNA sequences attached intandem. Hence, one promoter element can enhance the expression of one ormore recombinant products. Multiple promoter elements are well-known topersons of ordinary skill in the art.

The term “enhancers” or “enhancer” as used herein can refer to a DNAsequence that is located adjacent to the DNA sequence that encodes arecombinant product. Enhancer elements are typically located upstream ofa promoter element or can be located downstream of a coding DNA sequence(e.g., a DNA sequence transcribed or translated into a recombinantproduct or products). Hence, an enhancer element can be located 100 basepairs, 200 base pairs, or 300 or more base pairs upstream or downstreamof a DNA sequence that encodes recombinant product. Enhancer elementscan increase an amount of recombinant product expressed from a DNAsequence above increased expression afforded by a promoter element.Multiple enhancer elements are readily available to persons of ordinaryskill in the art.

The term “nuclear transfer” as used herein can refer to introducing afull complement of nuclear DNA from one cell to an enucleated cell.Nuclear transfer methods are well known to a person of ordinary skill inthe art. See, e.g., Nagashima et al., 1997, Mol. Reprod. Dev. 48:339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 73-78; Prather etal., 1989, Biol. Reprod. 41: 414-419; Prather et al., 1990, Exp. Zool.255: 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259:257-266; and Terlouw et al., 1992, Theriogenology 37: 309. Nucleartransfer may be accomplished by using oocytes that are not surrounded bya zona pellucida.

The terms “transfected” and “transfection” as used herein refer tomethods of delivering exogenous DNA into a cell. These methods involve avariety of techniques, such as treating cells with high concentrationsof salt, an electric field, liposomes, polycationic micelles, ordetergent, to render a host cell outer membrane or wall permeable tonucleic acid molecules of interest. These specified methods are notlimiting and the present disclosure relates to any transformationtechnique well known to a person of ordinary skill in the art.

The term “antibiotic” as used herein can refer to any molecule thatdecreases growth rates of a bacterium, yeast, fungi, mold, or othercontaminants in a cell culture. Antibiotics are optional components ofcell culture media. Examples of antibiotics are well known in the art.See Sigma and DIFCO catalogs.

The term “feeder cells” as used herein can refer to cells that aremaintained in culture and are co-cultured with target cells. Targetcells can be precursor cells, adipose stromal cells, cultured cells, andtotipotent cells, for example. Feeder cells can provide, for example,peptides, polypeptides, electrical signals, organic molecules (e.g.,steroids), nucleic acid molecules, growth factors (e.g., bFGF), otherfactors (e.g., cytokines such as LIF and steel factor), and metabolicnutrients to target cells. Certain cells may not require feeder cellsfor healthy growth. Feeder cells preferably grow in a mono-layer.

Feeder cells can be established from multiple cell types. Examples ofthese cell types are fetal cells, mouse cells, and oviductal cells.These examples are not meant to be limiting. Tissue samples can bebroken down to establish a feeder cell line by methods well known in theart (e.g., by using a blender). Feeder cells may originate from the sameor different animal species as precursor cells. Feeder cells can beestablished from fetal cells, mammalian fetal cells, and murine fetalcells.

The term “receptor ligand cocktail” as used herein can refer to amixture of one or more receptor ligands. A receptor ligand can refer toany molecule that binds to a receptor protein located on the outside orthe inside of a cell. Receptor ligands can be selected from molecules ofthe cytokine family of ligands, neurotrophin family of ligands, growthfactor family of ligands, and mitogen family of ligands, all of whichare well known to a person of ordinary skill in the art. Examples ofreceptor/ligand pairs are: vascular endothelial growth factorreceptor/vascular endothelial growth factor, epidermal growth factorreceptor/epidermal growth factor, insulin receptor/insulin,cAMP-dependent protein kinase/cAMP, growth hormone receptor/growthhormone, and steroid receptor/steroid. It has been shown that certainreceptors exhibit cross-reactivity. For example, heterologous receptors,such as insulin-like growth factor receptor 1 (IGFR1) and insulin-likegrowth factor receptor 2 (IGFR2) can both bind IGF1. When a receptorligand cocktail comprises a stimulus, the receptor ligand cocktail canbe introduced to a precursor cell in a variety of manners known to aperson of ordinary skill in the art.

The term “cytokine” as used herein can refer to a large family ofreceptor ligands well-known to a person of ordinary skill in the art.The cytokine family of receptor ligands includes such members asleukemia inhibitor factor (LIF); cardiotrophin 1 (CT-1); ciliaryneurotrophic factor (CNTF); stem cell factor (SCF), which is also knownas Steel factor; oncostatin M (OSM); and any member of the interleukin(IL) family, including IL-6, IL-1, and IL-12. The teachings of thepresent disclosure do not require the mechanical addition of steelfactor (also known as stem cell factor in the art) for the conversion ofprecursor cells into totipotent cells.

II. Methods of Isolating Autologous Stem Cells from Adipose Tissues

In accordance with the present disclosure, it has been discovered thattherapeutic quantities of autologous stem cells may be obtained fromadipose tissue. Such stem cells can be obtained as described herein.

For example, adipose tissue is obtained from an animal, preferable ahuman, and most preferably from the patient who is the intendedrecipient of the therapeutic stem cells. The tissue may be obtained bynumerous methods known in the art, including without limitationliposuction, and surgery. Preferably, the adipose tissue is obtained byliposuction, which is a simple, minimally invasive procedure.

In an exemplary method, following collection, adipose tissue isoptionally washed, for example with saline (such as PBS) to removedloose matter. Next, the tissue is digested with an enzyme, (such ascollagenase, dispase, or trypsin), and/or may also be degraded bymechanical agitation, sonic energy, thermal energy, and the like. Theresultant product is then optionally filtered, and then centrifuged toseparate the stromal cells from the adipose cells. Another round ofwashing and centrifugation may be performed in order to further purifythe cells. The cells may also be sorted using flow cytometry or othercell sorting means, and further isolated. Cells so obtained exhibit stemcell markers, eg. CD34.

Other methods of isolating stem cells from adipose tissue are known inthe art, and are disclosed for example in Zuk P. A. et al., TissueEngineering (2001) 7:211-228).

III. Methods of Inducing Stem Cells to Differentiate and/or ExpressGrowth Factors

Stem cells derived from adipose tissue as set forth above may be used inmany therapeutic procedures. Transgenic stem cells so obtained may alsobe optionally transfected at this point with a “corrective nucleic acidsequence”. The stem cells so obtained are then passaged and exposed to areceptor ligand cocktail to induce differentiation into the desired celllineage as exemplified herein below.

Tissues currently being developed from stem cells include, but are notlimited to: blood vessels (Kocher A. A. et al., Nature Med. (2001)7:430-436; Jackson K. A. et al., J. Clin. Invest. (2001) 107:1395-1402),bone (Petite H. et al., Nature Biotech. (2000) 18:959-963), cartilage(Johnstone B. et al., Clin. Orthop. (1999) 5156-5162), cornea (Tsai R.J. et al., N. Eng. J. Med. (2000) 343:86-93), dentin (Gronthos S. etal., Proc. Natl. Acad. Sci. USA (2000) 97:13625-13630), heart muscle(Klug M. G. et al., J. Clin. Invest. (1996) 98:216-224; review BohelerK. R. et al., Cir. Res. (2002) 91:189-201), liver (Lagasse E. et al.,Nature Med. (2000) 6:1229-1234), pancreas (Soria B. et al., Diabetes(2000) 49:1-6; Ramiya V. K. et al., Nature Med. (2000) 6:278-282),nervous tissue (Bjorkland A., Novartis Found. Symp. (2000) 231:7-15; LeeS. H. et al., Nature Biotechnology (2000) 18:675-679; Kim J. H. et al.,Nature (2002) 418:50-56), skeletal muscle (Gussoni E. et al., Nature(1999) 401:390-394), and skin (Pellegrini G. et al., Transplantation(1999) 68:868-879). Some of the tissues being generated from stem cellsare described in further detail below.

Heart Muscle

The loss of cardiomyocytes from adult mammalian hearts is irreversibleand leads to diminished heart function. Methods have been developed inwhich embryonic stem (ES) cells are employed as a renewable source ofdonor cardiomyocytes for cardiac engraftment (Klug, M. G. et al., J.Clin. Invest. (1996) 98:216-224). The ASC cells of the presentdisclosure can be similarly differentiated.

ES cells were first transfected by electroporation with a plasmidexpressing the neomycin resistance gene from an .alpha.-cardiac myosinheavy chain promoter and expressing the hygromycin resistance gene underthe control of the phosphoglycerate kinase (pGK) promoter. A proportionof cells comprising an activatable .alpha.-cardiac myosin heavy chainpromoter will differentiate into cardiac cells. Transfected clones wereselected by growth in the presence of hygromycin (200 μg/ml;Calbiochem-Novabiochem). Transfected ES cells were maintained in theundifferentiated state by culturing in high glucose DMEM containing 10%fetal bovine serum (FBS), 1% nonessential amino acids, and 0.1 mM2-mercaptoethanol. The medium was supplemented to a final concentrationof 100 U/ml with conditioned medium containing recombinant LIF.

To induce differentiation, 2×10⁶ freshly dissociated transfected EScells were plated onto a 100-mm bacterial Petri dish containing 10 ml ofDMEM lacking supplemental LIF. Regions of cardiogenesis were readilyidentified by the presence of spontaneous contractile activity. Forcardiomyocyte selection, the differentiated cultures were grown for 8days in the presence of G418 (200 μg/ml; GIBCO/BRL). Cultures ofselected ES-derived cardiomyocytes were digested with trypsin and theresulting single cell preparation was washed three times with DMEM anddirectly injected into the ventricular myocardium of adult mice.

The culture obtained by this method after G418 selection is more 99%pure for cardiomyocytes based on immunofluorescence for myosin. Theobtained cardiomyocytes contained well defined myofibers andintercalated discs were observed to couple juxtaposed cells consistentwith the observation that adjacent cells exhibit synchronous contractileactivity.

Importantly, the selected cardiomyocytes were capable of forming stableintercardiac grafts with the engrafted cells aligned and tightlyjuxtaposed with host cardiomyocytes. As mentioned previously the adiposestromal cells of the present disclosure can be similarly treated togenerate cardiomyocytes.

Endothelial Cells

Endothelial cells are critical to neo-vascularization, and are known tosecrete numerous growth factors which promote healing and expansion.Administration of endothelial cells promotes angiogenesis, resulting inincreased vascularization and development. Methods of inducing stemcells to differentiate to endothelial cells are generally known in theart, and are disclosed for example, in Balconi et al., ArteriosclerThromb Vasc Biol. (2000) 20:1443-1451.

Stem cell lines were grown in the undifferentiated state either ongelatin (0.1%)-coated Petri dishes (CJ7) or on a feeder layer of STOmurine fibroblasts.

To initiate cell differentiation cells were briefly trypsinized andsuspended in Iscove's modified Dulbecco's medium with 15% FBS, 10 mg/mLinsulin (Sigma), 100 U/mL penicillin, 100 mg/mL streptomycin, and 450mmol/L monothioglycerol. A growth factor cocktail was added to theculture medium to optimize vascular differentiation and included:recombinant human VEGF (Peprotech Inc) at 50 ng/mL; recombinant humanerythropoietin (Cilag AG), at 2 U/mL; human bFGF (Genzyme), at 100ng/mL; and murine interleukin 6 (Genzyme), at 10 ng/mL.

Cells were seeded in bacteriological Petri dishes (1.5×10⁴ cells per35-mm Petri dish) and cultured for 11 days, without further feeding, at37° C. in an incubator with 5% CO₂ in air and 95% relative humidity. Thecells were routinely examined for the presence of endothelium-likestructures by whole-mount preparation, by using rat mAb MEC 7.46directly against mouse PECAM as primary antibody and commercial rabbitimmunoglobulins to rat immunoglobulin (DAKO) as a secondary antibody.

In some cases, endothelial cells were selected with the use of sheepanti-mouse CD31 mAb-coated magnetic beads (Dynabeads, Dynal AS). In someexperiments, endothelial cell lines could be obtained withoutimmunoselection. In those cases, after EB disaggregation, the cells wereimmortalized by PmT. In previous studies, it was observed that PmTspecifically immortalizes endothelial cells and not any other cell type.

A suitable culture medium for stem-cell derived endothelial cells isDMEM with 20% FBS, supplemented with 2 mmol/L glutamine, 100 U/mLpenicillin, 100 mg/mL streptomycin, 50 mg/mL endothelial cell growthsupplement (Sigma), and 100 mg/mL heparin (Sigma) (Balconi et al.,Arterioscler Thromb Vasc Biol. (2000) 20:1443-1451).

ASCs in accordance with the present disclosure can be induced to formendothelial cells in a similar fashion.

Neuronal Cells

Parkinson's disease is caused by the loss of midbrain neurons thatsynthesize the neurotransmitter dopamine. Delivery ofdopamine-synthesizing neurons to the midbrain should alleviate thesymptoms of the disease by restoring dopamine production. Stem cellsobtained using the methods of the present disclosure may bedifferentiated into dopamine-synthesizing neurons utilizing theexemplary protocols set forth below. (Lee, S. H. et al., NatureBiotechnology, (2000) 18:675-679; Kim, J. H. et al., Nature (2002)418:50-56).

In a murine model, mouse ES cells were first transfected byelectroporation with a plasmid expressing nuclear receptor related-1(Nurr1), a transcription factor that has a role in the differentiationof midbrain precursors into dopamine neurons and a plasmid encodingneomycin resistance. Transfected clones (Nurr1 ES cells) were thensubsequently isolated by culturing the cells in G418. The Nurr1 ES cellswere then expanded under cultures which prevented differentiation (e.g.,growth on gelatin-coated tissue culture plates in the presence of 1,400U/ml-I of leukemia inhibitory factor (LIF; GIBCO/BRL, Grand Island,N.Y.) in ES cell medium consisting of knockout Dulbecco's minimalessential medium (GIBCO/BRL) supplemented with 15% FCS, 100 mM MEMnonessential amino acids, 0.55 mM 2-mercaptoethanol, L-glutamine, andantibiotics (all from GIBCO/BRL)). Selection of nestin-positive cells, amarker of developmental neurons, was initiated by replacing the ES cellmedium by serum-free Dulbecco's modified Eagle's medium (DMEM)/F12 (1:1)supplemented with insulin (5 μg/ml), transferrin (50 μg/ml), seleniumchloride (30 nM), and fibronectin (5 μg/ml) (ITSFn) medium. After 6-10days of selection, expansion of nestin-positive cells was initiated.Specifically, the cells were dissociated by 0.05% trypsin/0.04% EDTA,and plated on tissue culture plastic or glass coverslips at aconcentration of 1.5-2×10⁵ cells/cm2 in N2 medium modified (described inJohe K. et al., Genes Dev. (1996) 10:3129-3140), and supplemented with 1μg/ml of laminin and 10 ng/ml of bFGF (R&D Systems, Minneapolis, Minn.)in the presence of murine N-terminal fragment of sonic hedgehog (SHH;500 ng/ml) and murine fibroblast growth factor (FGF) 8 isoform b (100ng/ml; both from R&D Systems). Before cell plating, dishes andcoverslips were precoated with polyornithine (15 mg/ml) and laminin (1μg/ml, both from Becton Dickinson Labware, Bedford, Mass.).Nestin-positive cells were again expanded for six days. The medium waschanged every two days. Differentiation was induced by removal of basicFGF (bFGF). The differentiation medium consisted of N2 mediumsupplemented with laminin (1 mg/ml) in the presence of cAMP (1 μM) andascorbic acid (200 μM, both from Sigma St. Louis, MC). The cells wereincubated under differentiation conditions for 6-15 days.

78% of Nurr1 ES cells were found to be induced intodopamine-synthesizing, tyrosine hydroxylase (TH, 4 rate limiting enzymein the biosynthesis of dopamine) positive neurons by the method setforth above. The resultant neurons were further characterized to expressa variety of midbrain-specific markers such as Ptx3 and Engrailed 1(En-1). The dopamine-synthesizing, TH+ cells were also grafted into arodent model of Parkinson's disease and were shown to extend axons, formfunctional synaptic connections, perform electrophysiological functionsexpected of neurons, innervate the striatum, and improve motorasymmetry.

The ASCs of the present disclosure may be induced to form neuronal cellsin a similar fashion.

Insulin-Producing Cells

An ideal treatment for diabetes is the restoration of β-cell function ormimicking the insulin secretory pattern of these cells.Insulin-secreting cells derived from stem cells have been generated bythe following method and have been shown to be capable of normalizingblood glucose levels in a diabetic mouse model (Soria, B. 10 et al.,Diabetes (2000) 49:1-6).

ES cells were transfected by electroporation with a plasmid expressingβ-gal under the control of the human insulin regulatory region andexpressing the hygromycin resistance gene under the control of the pGKpromoter. Transfected clones were selected by growth in the presence ofhygromycin (200 pg/ml; Calbiochem-Novabiochem). Transfected ES cellswere maintained in the undifferentiated state by culturing in highglucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetalbovine serum (FBS), 1% nonessential amino acids, 0.1 mM2-mercaptoethanol, 1 mM sodium pyruvate, 100 IU/ml penicillin, and 0.1mg/ml streptomycin. The medium was supplemented to a final concentrationof 100 U/ml with conditioned medium containing recombinant LIF.

To induce differentiation to an insulin-secreting cell line, 2×10⁶hygromycin-resistant ES cells were plated onto a 100-mm bacterial Petridish and cultured in DMEM lacking supplemental LIF. For ES Ins/β-galselection, the differentiated cultures were grown in the same medium inthe presence of 200 μg/ml G418. For final differentiation andmaturation, the resulting clones were trypsinized and plated on a 100-mmbacterial Petri dish and grown for 14 days in DMEM supplemented with 200μg/ml G418 and 10 mM nicotinamide (Sigma), a form of Vitamin B3 that maypreserve and improve beta cell function. Finally, the resulting clusterswere cultured for 5 days in RPMI 1640 media supplemented with 10% FBS,10 mM nicotinamide, 200 μg/ml G418, 100 IU/ml penicillin, 0.1 mg/mlstreptomycin, and low glucose (5.6 mM).

For cell implantation, ES-derived insulin-secreting cells were washedand resuspended in RPMI 1640 media supplemented with 10% FBS, 10 mMnicotinamide, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 5.6 mMglucose at 5×10⁶ cells/ml. The mice to receive the implantation ofES-derived insulin-secreting cells were male Swiss albino mice that haddiabetic conditions induced by a single intraperitoneal injection ofstreptozotocin (STZ, Sigma) at 200 mg/kg body weight in citrate buffer.1×10⁶ cells were injected into the spleen of mice under anesthesia.

The ES-derived insulin-secreting cells produced from this methodproduced a similar profile of insulin production in response toincreasing levels of glucose to that observed in mouse pancreaticislets. Significantly, implantation of the ES-derived insulin-secretingcells led to the correction of the hyperglycemia within the diabeticmouse, minimized the weight loss experienced by the mice injected withSTZ, and lowered glucose levels after meal challenges and glucosechallenges better than untreated diabetic mice and similar to controlnondiabetic mice.

The ASCs of the present disclosure may be similarly differentiated intoinsulin producing cells using similar methods.

Hematopoietic Cells

Hematopoietic cells are used in numerous therapeutic regimens such asreplenishing bone marrow supply, or treating hematological disorders,such as anemias, thalassemias, and so forth. Stem cells can bedifferentiated into hematopoietic stem cells by methods known in theart, and disclosed, for example, in Howell et al., ExperimentalHematology (2002) 30:915-924.

In Howell et al., CD45−Sca-1+c-kit-cells were incubated for nine days inMEM+10% HS in the presence of 5 ng/mL mSCF, mFlt-3L, and MGDF, which arecytokines. Cells were then expanded. Cells expressed increased levels ofc-kit.

The in vivo repopulating potential of these cells was then assessed bytransplanting 4×10⁴ cells into lethally irradiated Boy J (CD45.1)recipient mice. Analysis of peripheral blood after 4 months revealedthat freshly isolated cells exhibited 7.9%+/−2.9% chimerism in recipientmice. A mathematical derivation of fold expansion of engraftmentpotential of cultured cells, relative to fresh cells revealed that thesecytokine cultured cells have a 5 fold increase in relative hematopoieticrepopulating potential.

The ASCs of the present disclosure may be similarly differentiated intohematopoietic cells, and used therapeutically to treat hematopoieticdisorders.

The recently described ability to genetically manipulate human ES cellsshould allow for the rapid isolation of highly uniform and singularlydifferentiated cells (Eiges R. et al., Current Biol. (2001) 11:514-518).A potential method to this end would be to employ a similar method tothat described above for murine ES cells in which antibiotic resistancegenes or selectable marker genes are expressed under cell specificpromoters. Alternatively, cell-type specific transcription factors orany other cell-type specific factors found to drive cell-type specificdifferentiation can be expressed.

IV. Administration of Adipose Derived Stem Cells for Therapeutic Benefit

The adipose derived stem cells of the present disclosure can be used ina wide array of therapeutic procedures. These therapies includeadministration of adipose stem cells, administration of stem cells whichsecrete growth factors such as VEGF, and stem cells which have beentransfected to express a desired therapeutic molecule.

These molecules may be administered by a variety of methods, includingretrograde coronary venous delivery, direct injection, arterial infusionvia catheter, systemic administration via IV, and so forth.

Methods of delivery of molecules to the heart via retrograde coronaryvenous delivery are generally known in the art and are described forexample in Boekstegers P. et al., Gene Ther (2000) 7:232-240, Herity N.A., Cathet. Cardiovasc. Intervent. (2000) 51:358-363, Suzuki et al.,Circulation (2000) III:359-364, and Yock et al., U.S. Pat. No.6,346,098. The injection of cells via RCVD can be at rates which achievepressures of about 30-400 mm Hg, most preferably about 50-150 mm Hg.

Retrograde coronary venous delivery is a suitable method for delivery ofother types of stem cells having therapeutic benefit. Such stem cellsinclude without limitation bone marrow derived stem cells, peripheralblood mononuclear derived stem cells, and skeletal tissue derivedmyocytes.

Methods of direct injection comprise primarily direct infusion, ideallyby hypodermic needle to the site where increased vascularization isdesired. For example, adipose stem cells of the present disclosure maybe administered via intramuscular injection, to a tissue whereangiogenesis is desired.

Methods of infusion via indwelling catheter generally comprise placementof an indwelling catheter to a region where stem cell therapy would bebeneficial, and administration of the adipose stem cells of the presentdisclosure through such a catheter.

The following non-limiting examples are provided to further illustratethe present disclosure.

EXAMPLE 1

Isolation and Characterization of Stem Cells from Adipose Tissue

Isolation of Stem Cells from Adipose Tissue

To isolate adipose stromal cells (ASCs,) subcutaneous adipose tissue(5-15 grams) was obtained from obese patients undergoing a gastroplastyprocedure. The tissue was suspended in PBS and dissected into smallerpieces using a scalpel. Collagenase (Worthington Biochemical) was thenadded to the suspension and placed in a shaker at 37° C. forapproximately 90 minutes. The digested sample was filtered through a 750micron Nitex filter and a 50 micron filter, and rinsed with Dulbecco'sModified Eagle Media (DMEM) with 10% Fetal Bovine Serum (FBS). Thefiltered suspension was centrifuged at 200 g for 5 minutes and the toplayer consisting of adipocytes was discarded. After a second spin at 300g for 5 minutes, any remaining adipocytes in the top layer were againdiscarded. The cell pellet was re-suspended in red cell lysis buffer andcentrifuged at 300 g after 5-10 minutes of incubation at 37° C. Thiscell pellet consisted of adipose stromal cells and was re-suspended inthe desired media. Adipose stromal cells (ASCs) were plated on tissueculture flasks at densities ranging from 1000 to 10,000 cells per cm².Non-adherent cells were discarded on the following day. The majority ofASCs attach to the flask and these adherent cells (FIG. 1) can beexpanded in either DMEM media with 10% FBS or in EGM2MV media(Clonetics), which contains the growth factors VEGF, bFGF, EGF, IGF and5% FBS. Cells cultured in EGM2MV media, for example, can be expanded50-fold in 8 days (FIG. 2) with their growth rate decreasing when theyreach confluency. ASCs continue to have a high proliferative activitywhen they are passaged. ASCs were also isolated from porcine and murineadipose tissue using similar isolation and culture protocols.

These experiments demonstrate that ASCs can be readily isolated andrapidly expanded ex vivo from relatively small amounts of adiposetissue, thus indicating that autologous ASCs may be used to advantage inresearch and clinical protocols. Human cardiovascular cell therapystudies suggest that 10⁸ to 10⁹ autologous cells may be required forclinical applications.

Considering the fact that liposuction can yield up to 3 liters ofsubcutaneous fat tissue in a single outpatient procedure, such cellnumbers can be easily obtained from patients with little or noexpansion.

Expression of Stem Cell Markers on ASCs

To evaluate the expression of the stem cell marker CD-34, adiposestromal cells were freshly isolated from human adipose tissue andlabeled with fluorescent antibodies against CD34 (BD Biosciences). As CD34 is also expressed on mature endothelial cells and endothelialprogenitor cells, the isolated cells were co-labeled with an antibodydirected against human VE-cadherin (BD Biosciences), a highly specificmarker of endothelial cells (Rafii S., J Clin Invest. (2000) 105:17-19).Data from a representative experiment is shown in FIG. 3. The resultsindicate that the majority of cells are CD34+/VE-cadherin−, thussuggesting that most human adipose tissue derived stromal cells are nonendothelial CD34-positive stem or progenitor cells. The human adiposestromal cell fraction also contains a small but distinct population ofCD34+/VE-cadherin+ cells, which may represent either mature endothelialcells or endothelial progenitor cells.

To determine whether the expression of stem cell markers could also befound on adipose stromal cells in the mouse, murine ASCs were isolatedfrom mice using the same protocol that was used for human ASC isolation.Murine ASCs were cultured, expanded in DMEM/F12 media with 10% fetalbovine serum and passaged when confluent. The passaged cells werefinally detached with EDTA at confluence and subsequently labeled withan antibody directed against the specific murine stem cell marker Sca-1(Stem Cell Antigen-1). As shown in FIG. 4, greater than 70% of culturedmurine ASCs express the stem cell marker Sca-1. The high level ofexpression of CD34 in human ASCs and of Sca-1 on murine ASCs complementsthe data on ASC differentiation potential (Zuk P. A. et al., TissueEngineering (2001) 7:211-228; Erickson et al., Biochem Biophys ResCommun. (2002) 290:763-769; Safford K. M. et al., Biochem Biophys ResCommun. (2002) 294:371-379) and provides evidence that ASCs have stemcell characteristics.

EXAMPLE II

Differentiation of Adipose Derived Stromal Cells

ASCs can Develop a Vascular Phenotype

A major pathway by which ASCs can enhance angiogenesis is bydifferentiation of ASCs into a vascular cell phenotype. Since ASCs arealready known to differentiate into a number of cell types like muscle,bone and neural cells, it was evaluated whether ASCs can developphenotypes that correspond to either vascular endothelial cells orvascular smooth muscle cells. Plated adherent human ASCs were evaluatedby flow cytometry for the expression of the stem cell marker CD34 andthe endothelial marker VE-Cadherin, (FIG. 5). While the majority ofcells are still CD34+/VE-Cadherin-, there has emerged a substantialproportion of cells that are CD34+/VE-Cadherin+; this finding has beenreplicated consistently with samples from several donors. This is incontrast to freshly isolated ASCs, which express minimal VE-Cadherin(FIG. 3), prior to plating. The appearance of a CD34+/VE-Cadherin+ cellpopulation suggests that differentiation towards an endothelialphenotype may have occurred. This experiment also demonstrates that theadherent ASC population continues to express high levels of the stemcell marker CD34 both in conjunction with the expression of VE-cadherin,as well as in a VE-cadherin-negative population. In parallel experiments(data not shown), it was found the human ASC population began to expressan additional endothelial marker, the VEGF receptor-2, (KDR or flk-1)following passage, whereas they did not express this marker atdetectable levels when freshly isolated; this finding supports theinterpretation of direction towards an endothelial lineage.

To examine whether ASCs can also manifest typical endothelial behaviorin vitro, their phenotype on Matrigel was determined. Mature endothelialcells when plated overnight on Matrigel (BD Biosciences) form tube- andcord-like structures, reminiscent of a capillary network. Humansubcutaneous ASCs that had been cultured in EGM2MV media on Matrigelovernight were plated, and the non-adherent cells were discarded thefollowing day. Human aortic endothelial cells were also plated as apositive control in a separate Matrigel well (FIG. 6). The endothelialcells formed the expected tube and cord-like structures. Interestingly,ASCs also formed similar structures, further supporting the potential ofASCs to develop an endothelial phenotype.

In parallel studies designed to evaluate whether ASCs could also developa smooth muscle phenotype, we stained ASCs cultured in DMEM/10% FBSmedia for the smooth muscle-specific marker, smooth muscle alpha-actin(FIG. 7). After culture in DMEM/10% FBS, roughly 40-50% of ASCs stainpositive for alpha-actin, suggesting the development of a smooth muscleor myofibroblast phenotype. ASCs cultured in EGM2MV show essentially nocells staining positively for SM-alpha actin.

Together, these data suggest that ASCs can develop phenotypes ofspecific vascular cells, in a fashion that is directly responsive totheir growth factor and matrix environments, suggesting a pliability ofphenotype in these cells. These data demonstrate that ASCs may be ableto contribute to angiogenesis or arteriogenesis directly by providingdifferentiated cells for incorporation into nascent vascular structures.

EXAMPLE III

Secretion of Growth Factors by Adipose Derived Stromal Cells

Secretion of Angiogenic Growth Factors by ASCs

Since stem and progenitor cells can secrete multiple growth factors(Majka M. et al., Blood (2001) 97:3075-3085) and putative therapeuticutilities could in part be related to this endocrine function, thesecretion of angiogenic growth factors by human subcutaneous ASCs wasevaluated. Human subcutaneous adipose stromal cells were cultured inEGM-2-MV media to confluence, and then switched into growth-factor freebasal media (EBM-2, Clonetics) for 72 hours. Cell supernatants werecollected and subsequently assayed for angiogenic growth factors usingELISA and Multi-Analyte-Profiling kits (R&D Systems) for VascularEndothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF) andGranulocyte-Colony Stimulating Factor (G-CSF). The cell number wasassessed at the end of the 72-hour period, and the secretion of growthfactors is expressed in pg/10⁶ ASCs. The cell supernatants containedsignificant amounts of secreted VEGF, HGF and G-CSF (FIG. 8). On theother hand, subcutaneous ASCs did not secrete detectable levels of thearteriogenic growth factor basic FGF. These findings indicate that inaddition to the pluripotency of ASCs, their endocrine or paracrinepotential may have significant therapeutic relevance; ASCs delivered tothe heart in the setting of coronary or peripheral arterial occlusivedisease, for example, may be able enhance angiogenesis not only bydifferentiating into a vascular phenotype, but also by recruitingresident mature vascular endothelial cells to integrate into the nascentvascular network.

EXAMPLE IV

Stromal Cells Can be Transfected with Mammalian Plasmids and Used forGene Therapy

Transfection of Adipose Derived Stromal Cells with Green-FluorescentProtein (GFP)

ASCs are suitable host cells for transfection by mammalian expressionplasmids, thus allowing for use of ASCs as cellular “vectors” for genetherapy. Such transfected cells should supplement the therapeuticeffects of endogenously secreted growth factors. Porcine ASCs wereisolated from subcutaneous porcine adipose tissue and cultured in EGM2MVmedia. Confluent ASC cultures at passages 0 through 4 were suspended inPBS and electroporated with green fluorescent protein (GFP) plasmid.Optimal conditions to achieve high levels of transfection were anelectroporation voltage of 300V, capacitance of 9601 μF and a cellconcentration of 5×10⁴ to 1.5×10⁵ cells per μg of DNA. Electroporatedcells were plated and GFP expression was assessed on days 1-3 posttransfection. Transfection efficiency as assessed by either manualcounting of GFP-positive cells (FIG. 9) or by flow cytometric analysisof positive cells was routinely 50% or higher. These data show that ASCscan be transfected with non-viral methods and therefore be used asautologous cell vectors for gene therapy. This application of ASCs willbe particularly beneficial in the therapeutic setting using plasmids,which encode exogenously secreted factors, which complement the activityof endogenously produced factors.

EXAMPLE V

Further Characterization of Adipose Stromal Cells

Flow Cytometric Comparison of Murine ASCs and Muscle Derived Stem Cells

Previous data indicated that the expression of Sca-1 on murine ASCs, andtherefore suggested similarity between the subcutaneous ASC populationand muscle derived Sca-1(+)CD45(−)c-kit(−) stem cells. The phenotype ofmurine ASCs was further defined by co-staining for the markers CD45 andc-kit. As shown in FIG. 10, murine ASCs contain a significantSca-1(+)CD45(−)c-kit(−) cell population. These findings support andextend the data showing the similarity of murine ASCs and the musclederived stem cells. This in turn provides further evidence that ASCs mayhave a hematopoietic potential similar to what has been shown formuscle-derived stem cells (Howell J. et al., Exp. Hem. 30:915-924,2002).

EXAMPLE VI

Further Differentiation of Adipose Derived Stromal Cells

Differentiation of Sca-1(+)CD45(−)c-kit(−) Cells

To characterize the pluripotentiality of adipose Sca-1(+)CD45(−)c-kit(−)cells in direct comparison to that of muscle derived stem cells,Sca-1(+)CD45(−)c-kit(−) cells were purified from both tissues by cellsorting, and cultured in either neuronal or adipose differentiationmedia.

By day 12, muscle-derived Sca-1(+)CD45(−)c-kit(−) cells differentiatedinto adipocyte-like (with IGF-1) or neuron-like (with bFGF and PDGF)cells (FIGS. 11A and B). Neuronal differentiation of these cells wasconfirmed by positive staining for the tau-protein (FIG. 11C.) Thesimilarly purified population of adipose-derived Sca-1(+)CD45(−)c-kit(−)cells also responded to these respective growth conditions bydifferentiating into adipocyte-like (FIG. 11D) and neuron-like cells(FIG. 11E). Staining for tau-protein for adipose-derived cells inneuronal differentiation media was not yet available at the time.Control experiments with Sca-1-negative cells did not demonstrate anysignificant transdifferentiation, thus showing for the first time thatpluripotency of ASCs may be specifically observed in a particular sortedpopulation. These data suggest that Sca-1(+)CD45(−)c-kit(−) cells withinthe uniquely accessible adipose tissue compartment, have comparabledifferentiation potential to similar cells derived from skeletal muscle,and thus may represent a common stem cell in multiple tissues.

EXAMPLE VII

Adipose Stromal Cells can be Delivered to the Myocardium Via RetrogradeCoronary Venous Delivery (RCVD) and Direct Injection

Delivery of ASCs to the Myocardium of Pigs Via RCVD

Juvenile farm pigs, 25-35 kg, were used. The anterior interventricularvein (AIV) or posterior vein of left ventricle (PVLV) was cannulated bya balloon-tipped catheter. The balloon was inflated to prevent venousregurgitation and myocardial blush was confirmed by 1.0-1.5 cc contrastinjection.

Human adipose stromal cells, and porcine aortic endothelial cells weredelivered via retrograde coronary venous delivery (RCVD.) Briefly, 5 mlof 2×10⁶ pre-labeled human adipose stromal cells or porcine aorticendothelial cells were labeled with either PKH26 or BrdU, andadministered to pigs. Animals were euthanized 1-1.5 hours post delivery.

The heart was then sliced transversely, perpendicular to theapical-basal axis. Planimetry measurement was performed using NIH Image.Cells in the area of myocardial blush were evaluated by fluorescencemicroscopy or immunostaining for BrdU (See FIGS. 12A-D.)

Retrograde infusion was well tolerated in all animals without adversehemodynamic effects; the arterial pressure and heart rate did not changesignificantly following delivery. The retrograde infusion time was5.6.±2.8 sec. Non-sustained ventricular tachycardia was limited to thetime of retrograde infusion, consisting of 5 to 8 ectopic beats andceasing immediately following the injection.

As shown in FIGS. 12A-D, PKH26 and BrdU pre-labeled cells were observedat the target myocardium tissue. From this example, it is clear thatmultiple cell types can be delivered into the myocardium using RCVD, andthat RCVD results in widespread cell distribution in the myocardialtissue.

Delivery of ASCs to the Myocardium of Pigs via Direct Injection

Direct Injection of BrdU and PKH26 labeled cells was performed using atuberculine syringe. After the heart was exposed, 200 microliters ofBrdU pre-labeled cell suspension containing 2×10⁵ cells was injectedinto the left ventricle free wall, at nine discrete injection sites. SeeFIGS. 12E-F. Alternatively, cells can be injected into the ventricularmuscle using needle devices which are introduced from the ventricularlumen (injection into the endocardial surface), or into veins and/orarteries coursing over the heart. These techniques for intramuscular andintravascular injection are generally known in the art.

The foregoing results demonstrate that delivery of stem cells via RCVDgives rise to a wider distribution of cells in target tissue than directmicroinjection.

EXAMPLE VIII

An In vivo Model for Assessment of Angiogenesis

In vivo Assessment of Angiogenesis Using a Mouse Hindlimb Ischemia Model

An in vivo model of quantifying angiogenesis over time has beenestablished in a mouse model using hindlimb ischemia. This has allowedidentification of modulators of angiogenesis. In one study, for example,the role of the GTP-binding signalling molecule Rac2 was evaluated.

Wild-type mice (C57BL6) and Rac2−/−mice underwent ligation of thefemoral artery below the inguinal ligament.

Non-invasive laser doppler imaging (LDI) (Moor Instruments) wasperformed on the ischemic and non-ischemic hindlimbs of all mice onpost-operative days 1, 4, 7, 10, 14 and 21, in order to evaluate bloodflux as a surrogate for perfusion. A representative LDI image is shownin FIG. 13, where the Rac2−/− mouse has significant impairment of bloodflow to the ischemic leg (Panel 13A), manifested by a blue shift in thecolors of the pseudocolored pixels, while the wild-type mouse hasrecovered most of the perfusion to the ischemic leg, reflected by abilaterally symmetric flux reflected in the pseudocolor image.

When the blood perfusion is measured over time and quantitated as aratio of blood perfusion in the ischemic to the non-ischemic leg, itbecomes apparent that the impairment in recovery of limb perfusion inRac2−/− mice when compared to wild-type mice is first evident onpost-surgery day 4 (FIG. 14). This difference between the two groups ismaintained through day 21. Statistical analysis using a between grouprepeated measures ANOVA shows a highly significant between groupdifference. This repeated in vivo assessment of angiogenesis allows oneto not only distinguish between global angiogenesis in a control andtreatment group, but also to observe time-dependent changes. This modelsystem can be used to quantify the effects of ASC transplantation intoischemic mouse hindlimbs on time-dependent angiogenesis.

Intramuscular Injection of ASCs into Ischemic Hindlimbs ImprovesPerfusion

Human ASCs were isolated and cultured in the presence of endothelialgrowth media (EGM-2-MV, Clonetics) containing the growth factors VEGF,bFGF, EGF and IGF. NOD/SCID immune deficient mice (n=6, aged 8 months)underwent unilateral femoral artery ligation and received intramuscularinjection of either 4×10⁵ human ASCs per mouse (n=3) or media (n=3) intothe quadriceps, gastrocnemius and tibialis muscles of the ischemichindlimb on the subsequent day. Due to the advanced age of the mice andthe reduced immunologic competence, the endogenous angiogenesis responseof the mice was markedly blunted, thus resulting in some degree ofeither toe or limb necrosis in all animals. However, by day 10 of thestudy, mice receiving ASC injections had a remarkably mitigated extentof limb necrosis (FIG. 15). This reduction in necrosis was statisticallysignificant (FIG. 16).

This study highlights the in vivo angiogenic potential of the cells. Anongoing hindlimb ischemia experiment with younger NOD/SCID mice has notresulted in any significant toe or limb necrosis, thus allowing us touse Laser Doppler Imaging to detect subtle perfusion differences betweencell-treated and media-treated animals.

The cells disclosed herein may optionally be administered by retrogradecoronary venous delivery, or other means set forth above.

In conclusion, these preliminary data support the hypotheses of ouroriginal proposal that ASCs have a phenotype and pluripotentiality thatis similar to that of muscle-derived stem cells and that they canaccordingly be used for cell therapies targeted at enhancingangiogenesis and hematopoiesis. The key advantage of identifying suchcells in subcutaneous adipose tissue is that they can be harvested byliposuction and therefore increase the clinical feasibility ofautologous cell therapy.

The previous examples and description set forth certain embodiments ofthe present disclosure. It should be appreciated that not all componentsor method steps of a complete implementation of a practical system arenecessarily illustrated or described in detail. Rather, only thosecomponents or method steps necessary for a thorough understanding of thepresent disclosure have been illustrated and described in detail. Actualimplementations may utilize more steps or components or fewer steps orcomponents. Thus, while certain of the preferred embodiments of thepresent disclosure have been described and specifically exemplifiedabove, it is not intended that the present disclosure be limited to suchembodiments. Various modifications may be made thereto without departingfrom the scope and spirit of the present disclosure, as set forth in thefollowing claims.

1. A mixed population of cells, obtained by: a. isolating subcutaneousadipose tissue from a mammal; b. dissociating the isolated subcutaneousadipose tissue into a cell suspension; c. removing adipocytes from thecell suspension, resulting in isolated cells; and d. culturing theisolated cells in EGM-2-MV media such that a mixed population of cellscomprising a first population of CD34+/VE-cadherin− cells and a second/population of CD34+/VE-cadherin+ endothelial cells are obtained.
 2. Thecells of claim 1, wherein the culturing is in EGM-2-MV media onmatrigel.
 3. The cells of claim 1, wherein the mixed population of cellsare further cultured in the presence of 5-azacytidine.
 4. The cells ofclaim 1, wherein the mixed population of cells are further cultured inin EBM-2 media.
 5. The cells of claim 4, which secrete a factor selectedfrom the group consisting of pro-angiogenic factors, anti-apoptoticfactors, vasculoprotective factors, and cardioprotective factors.
 6. Thecells of claim 1, wherein the first population is a larger quantity thanthe second population.
 7. The cells of claim 1, wherein the dissociatingis performed using agitation, sonic energy, thermal energy, or acombination thereof.
 8. The cells of claim 1, wherein the secondpopulation comprises a population of cells having an endothelialphenotype.
 9. The cells of claim 1, wherein the dissociating isperformed using collagenase, dispase, trypsin, or a combination thereof.10. The cells of claim 1, further cultured in DMEM and 10% fetal bovineserum.
 11. A mixed population of cells, obtained by: isolatingsubcutaneous adipose tissue using liposuction or surgery; dissecting thesubcutaneous adipose tissue and dissociating said tissue into a cellsuspension; removing adipocytes from the cell suspension; isolatingcells from the cell suspension after removal of the adipocytes; andculturing the isolated cells in EGM-2-MV media such that a mixedpopulation of cells comprising a first population of CD34+/VE-cadherin−cells and a second population of CD34+/VE-cadherin+ endothelial cellsare obtained.
 12. The cells of claim 11, wherein the dissociating isperformed using agitation, sonic energy, thermal energy, or acombination thereof.
 13. The cells of claim 11, wherein the dissociatingis performed using collagenase, dispase, trypsin, or a combinationthereof.
 14. The cells of claim 11, wherein the cells comprise anexogenous nucleic acid selected from the group of nucleic acids encodingVEGF, bFGF, IGF1, IGF2, HGF, cardiotrophin, myotrophin, nitric oxidesynthase 1, nitric oxide synthase 2, nitric oxide synthase 3, fibroblastgrowth factor, pleotrophin, endothelin, and angiopoietin.
 15. The cellsof claim 11, wherein the second population comprises a population ofcells having an endothelial phenotype.
 16. The cells of claim 11,further comprising the step of culturing the mixed population of cellsin EBM-2 media.
 17. The cells of claim 16, wherein the mixed populationof cells secrete at least one factor selected from the group consistingof a vascular endothelial growth factor (VEGF), a hepatocyte growthfactor (HGF), granulocyte-macrophage colony stimulating factor (GM-CSF),and a granulocyte-colony stimulating factor (G-CSF).
 18. A mixedpopulation of cells, obtained by: a) isolating subcutaneous adiposetissue from a mammal; b) dissecting the subcutaneous adipose tissue; c)dissociating the dissected subcutaneous tissue into a cell suspension;d) removing adipocytes from the cell suspension; e) isolating cells fromthe cell suspension after removal of the adipocytes; f) culturing thecells from step e) in EGM-2-MV media; and g) culturing the cells fromstep f) such that a mixed population of cells comprising a firstpopulation of CD34+/VE-cadherin− cells and a second population ofCD34+/VE-cadherin+ endothelial cells are obtained.
 19. The cells ofclaim 18, wherein the mixed population of cells contain pluripotentcells capable of differentiating into another cell type.
 20. The cellsof claim 18, wherein the mixed population of cells includes a populationof cells having an endothelial phenotype.